The goal of this project is to strengthen the ability of x-ray absorption spectroscopy to characterize complex mixtures often found in biological samples. X-ray fluorescence is sensitive to the chemical state of an atom. Thus, x-ray absorption spectra from chemically distinct sites in a complex sample can be obtained by monitoring the x-ray fluorescence signal with high energy resolution. Progress in x-ray spectrographs, detectors, and sources now allows this experiment to be applied to dilute materials such as metals in enzymes. Construction of an x-ray spectrograph using spherically bent crystals and a high spatial resolution detector is proposed. The fluorescence spectra of model compounds for elements from sulfur through molybdenum will be investigated. From these emission spectra the best energies for "site-specific x-ray absorption spectroscopy" will be determined. This technology will be applied to differentiate Mn(II,III,IV) environments in catalase and photosystem II, Cu(I,II) sites in mixed-valence cytochrome oxidase, and Fe(ll,III) species in binuclear iron oxygenases, and eventually to catalytic intermediates. K-edge absorption spectra recorded with high resolution fluorescence detection can show features sharper than the natural line width. This advantage will be used to characterize the S, V, Fe and Mo sites of nitrogenase. The instrumentation and methods developed during this project should be generally applicable to characterization of trace elements in biology.